JP2010521193A - RNA antagonist compounds for inhibition of APO-B100 expression - Google Patents

Abstract

Short oligonucleotides directed to the Apo-B100 gene are provided for modulating the expression of Apo-B100. Methods of using these compounds for the modulation of Apo-B100 expression and for the treatment of diseases associated with Apo-B100 expression are provided. The oligonucleotide comprises deoxyribonucleosides and locked nucleic acids.

Description

The present invention relates to oligonucleotides directed against the Apo-B100 gene that are provided to regulate the expression of Apo-B100. Methods are provided for using these compounds for modulation of Apo-B100 expression and for the treatment of diseases associated with Apo-B100 expression. The oligonucleotide comprises deoxyribonucleosides and locked nucleic acids.

BACKGROUND OF THE INVENTION See the background section of WO2007 / 031081, which is incorporated herein by reference.

  To date, strategies aimed at inhibiting the function of apolipoprotein B have been limited to Lp (a) apheresis, antibodies, antibody fragments and ribozymes. Further, PCT publications WO 00/97662, WO 03/11887 and WO 2004/44181 disclose and claim antisense oligonucleotides with low biostability and / or low binding affinity.

  As a result, there continues to be a need for additional substances that can effectively antagonize the function of apolipoprotein B and consequently reduce plasma Lp (a) levels.

  The present invention regulates apolipoprotein B expression, ApB-100, including an effective locked nucleic acid (LNA) oligomeric compound, and inhibition of ApoB-48, another isoform of apolipoprotein B Its use in the method.

The present invention is an oligomeric compound (oligomer) consisting of a total of 10-15 nucleobases, wherein the nucleobase sequence of the compound is present in SEQ ID NO: 1, and the compound is at least 2 or at least 3. Compounds comprising nucleotide analogs are provided.

  The present invention provides an oligomeric compound comprising a total of 10-15 nucleobases, the nucleobase sequence corresponding to a contiguous subsequence present in SEQ ID NO: 1 and comprising at least 2 or at least 3 nucleotide analogs To do.

  The present invention provides a complex comprising a compound of the invention and at least one non-nucleotide or non-polynucleotide moiety covalently linked to said compound.

  The present invention provides a pharmaceutical composition comprising a compound or complex of the present invention and a pharmaceutically acceptable diluent, carrier or adjuvant.

  The present invention provides a compound or complex of the present invention for use as a medicament.

  The present invention provides the use of a compound or complex of the present invention for the manufacture of a medicament for the treatment of abnormal levels of Apo-B100 or a disease or condition associated therewith.

  The present invention relates to a compound of the invention or a complex of the invention for the treatment of abnormal levels of Apo-B100 or a disease or condition associated therewith, such as atherosclerosis, hypercholesterolemia or hyperlipidemia. A medicament comprising:

  The present invention suffers from a disease or condition selected from atherosclerosis, hypercholesterolemia and hyperlipidemia comprising the step of administering to a subject in need thereof a pharmaceutical composition or complex or medicament of the present invention. A method of treating a subject is provided.

  The present invention provides a pharmaceutical composition or complex or medicament according to the present invention to a subject, for example a subject suffering from a medical condition selected from the group consisting of atherosclerosis, hypercholesterolemia or hyperlipidemia. A method of down-regulating apolipoprotein B comprising the step of administering is provided.

  The present invention provides a method for downregulating apolipoprotein B (ApoB) mRNA in a cell expressing ApoB mRNA, comprising the step of administering to the cell a compound of the present invention to downregulate the ApoB mRNA. Provide a method.

  The present invention is a method of down-regulating apolipoprotein B (ApoB) protein in a cell expressing ApoB protein, comprising the step of administering to said cell the compound of the present invention to down-regulate said ApoB protein Provide a method.

Brief description of the figure
ApoB-100 mRNA expression was measured by qPCR normalized to the housekeeping gene GAPDH and shown relative to the saline group. Mice (n = 3 or 5) were administered for 3 consecutive days, sacrificed 24 hours after the last dose, and livers were isolated and analyzed. Serum cholesterol levels at the time of sacrifice (day 3) after administration of different length oligos. ApoB-100 mRNA expression was measured by qPCR normalized to the housekeeping gene GAPDH and shown relative to the saline group. Mice (n = 5) were administered once, sacrificed on different days after administration, and the liver was isolated and analyzed. Total serum cholesterol measured at the time of sacrifice (days 1, 3, 5, 6 and 8) using the ABX pentra kit, n = 5. ApoB-100 mRNA expression was measured by qPCR normalized to the housekeeping gene GAPDH and shown relative to the saline group. Mice (n = 5) were administered once, sacrificed on different days after administration, and the liver was isolated and analyzed. Total serum cholesterol measured at the time of sacrifice (days 1, 3, 5, 6, 8, and 16) using the ABX pentra kit, n = 5. ApoB-100 mRNA expression was measured by qPCR normalized to the housekeeping gene GAPDH and shown relative to the saline group. Mice (n = 5) were dosed with 1 or 5 mg / kg for 3 consecutive days, sacrificed 24 hours (day 3) after the last dose, and livers were isolated and analyzed. Serum total cholesterol measured at the time of sacrifice (day 3) using the ABX pentra kit, n = 5. 1, 2.5, or 5 mg / kg / dose of SEQ ID NO: 26, A: once a week (days 0, 7, 14, 21, 28, 35, 42) or B: once every two weeks (0, Day 14, 28 and 42) Serum total cholesterol expressed as a percentage of the control group (saline) after administration. All animals are sacrificed 1. 49 days after administration. Data are shown as mean ± SD, n = 7. Total cholesterol in A) non-HDL and B) HDL fractions separated from plasma by ultracentrifugation. C57BL / 6J mice were administered SEQ ID NO: 26 once a week at 1, 2.5 or 5 mg / kg, and plasma was sampled immediately before the next administration for 1 week after administration. Data show mean ± SD and are shown relative to the saline control group, n = 5. Total cholesterol in A) non-HDL and B) HDL fractions separated from plasma by ultracentrifugation. C57BL / 6J mice were administered SEQ ID NO: 26 once every two weeks at 1, 2.5 or 5 mg / kg, and plasma was sampled one or two weeks after administration. Data show mean ± SD and are shown relative to the saline control group, n = 5.

DETAILED DESCRIPTION OF THE INVENTION US Provisional Applications 60 / 896,419 and 60 / 977,409 are incorporated herein by reference.

Oligomer compounds The present invention relates to oligomers for use in inhibiting the function of nucleic acid molecules encoding apolipoprotein B (e.g. Apo-B100 and / or ApoB-48) that cause a reduction in the number of functional proteins produced. Compounds, particularly antisense oligonucleotides are employed.

  The present invention provides compositions and methods for modulating the expression of apolipoprotein B (Apo-B100 / Apo-B48). In particular, the present invention relates to oligonucleotide compounds spanning a specific motif targeting apolipoprotein B. These motifs are SEQ ID NOs 2-15, in particular SEQ ID NOs 5, 9 and 13. Also disclosed are specific designs of LNAs containing oligonucleotide compounds. Particularly preferred compounds are SEQ ID NOs: 17-40 and / or 41-49, especially SEQ ID NOs: 16, 17, 26 and 34. The compounds of the present invention are potent inhibitors of apolipoprotein mRNA and protein expression.

  As used herein, the term “target nucleic acid” refers to DNA encoding Apo-B100, RNA transcribed from such DNA (including pre-mRNA and mRNA and mRNA editing), and cDNA derived from such RNA. Is also included.

  “Target protein” is mammalian apolipoprotein B, preferably human apolipoprotein B. Since ApoB-100 and ApoB-48 both originate from the same genetic sequence, the oligomeric compounds of the present invention produce either or both forms of apolipoprotein B, as well as mRNA encoding ApoB-100, and Apo-B48. It will be appreciated that it can be used for down-regulation of both encoded RNA editing forms.

  As used herein, the term “gene” refers to genes containing exons, introns, non-coding 5 ′ and 3 ′ regions and regulatory elements, and all currently known variants and potential elucidations Means any further variants.

  As used herein, the term “mRNA” means one or more currently known mRNA transcripts of the targeted gene and any additional transcripts that may be identified.

  As used herein, the term “modulation” means either an increase (stimulation) or a decrease (inhibition) of gene expression. In the present invention, inhibition is a preferred form of gene expression regulation and mRNA is a preferred target.

  As used herein, the term “targeting” an antisense compound to a specific target nucleic acid refers to an antisense compound in a manner that allows it to bind to a target of interest and modulate its function. By providing an oligonucleotide to a cell, animal or human.

  The terms “oligomer compound”, “oligonucleotide”, “oligo”, and “oligonucleotide compound” that are interchangeable with the term “oligomer” are used herein to refer to an oligomer, ie, a nucleic acid polymer (eg, ribonucleic acid (RNA)). Or deoxyribonucleic acid (DNA)) or their nucleic acid analogs known in the art, preferably locked nucleic acids (LNA), or mixtures thereof. The term includes oligonucleotides composed of natural nucleobases, sugars and internucleoside (backbone) linkages, as well as oligonucleotides having non-natural portions that function similarly or have certain improved functions. Oligonucleotides that have been modified or substituted in whole or in part may have some desirable properties of such oligonucleotides, such as the ability to permeate cell membranes, good resistance to extracellular and intracellular nucleases, high affinity for nucleic acid targets Due to its sex and specificity, it is often preferred over the natural form. For example, with respect to the above properties, LNA analogs are particularly preferred. Thus, in a highly preferred embodiment, the terms “oligomer compound”, “oligonucleotide”, “oligo”, “oligomer”, and “oligonucleotide compound” of the present invention refer to nucleotide and nucleotide analog units, such as LNA units. A compound that is constructed in both to form between 10-15 (continuous) nucleotide / nucleotide analog polymerizable compounds (oligomers).

  The oligomeric compound is preferably an antisense oligomeric compound, also referred to as 'antisense oligonucleotide' and 'antisense inhibitor'.

  Antisense inhibitors are single stranded oligonucleotides. Single stranded oligonucleotides are preferably complementary to corresponding regions of the target nucleic acid.

  Usually, single-stranded 'antisense' oligonucleotides interact specifically with the mRNA of the target gene, causing, for example, targeted degradation of the mRNA via the RNaseH mechanism or preventing translation.

  The term “unit” is understood to be a monomer.

  The oligomeric compounds of the invention can hybridize to one or more apolipoprotein B messenger RNA and / or the sense or complementary strand of mammalian apolipoprotein B (Apo-B) DNA. NCBI accession number NM_000384 provides the mRNA sequence of human apolipoprotein B. In a preferred embodiment, the oligomeric compound of the present invention is highly capable of hybridizing to the human apolipoprotein encoded by the nucleic acid disclosed in NCBI Accession No. NM_000384, or its reverse complement, comprising the mRNA nucleic acid target derived from the human apolipoprotein. Is preferable.

  The term “at least [integer]” means the integer, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, Includes integers greater than or equal to 19, 20, 21 etc.

  In an interesting embodiment, the 3 ′ end of the compounds of the invention comprise a nucleotide rather than a nucleotide analog.

In preferred embodiments, the oligonucleotide hybridizes to a target nucleic acid, such as ApoB mRNA, and has a T _{m of} at least 30 ° C, such as at least 37 ° C, such as at least 40 ° C, at least 50 ° C, at least 55 ° C, or at least 60 ° C. Can form a double strand. In one aspect, T _m is between 37 ° C. and 80 ° C., such as between 50 and 70 ° C. In one aspect, the T _m is between 30 ° C and 40 ° C.

Measurements of T _m
A 3 μM solution of the compound in 10 mM sodium phosphate / 100 mM NaCl / 0.1 nM EDTA at pH 7.0 and its complement at a concentration of 3 μM in 10 mM sodium phosphate / 100 mM NaCl / 0.1 nM EDTA at pH 7.0 Mix with typical DNA or RNA oligonucleotide at 90 ° C for 1 minute and allow to cool to room temperature. The duplex melting curve is then determined by measuring the absorption at 260 nm in the range of 25 to 95 ° C. with a heating rate of 1 ° C./min. T _m is measured as the maximum of the first derivative of the melting curve.

  In one embodiment, the oligomeric compounds of the invention can target DNA encoding mammalian ApoB.

  The term “nucleobase” as used herein refers to both natural nucleotides, such as DNA and RNA nucleotides (units), and non-natural nucleotides referred to as nucleotide analogs (units).

Sequence The present invention is directed to an oligomeric compound consisting of 10-15, eg 11-15, 12-15, 13-15, in particular 12-14 nucleobases, the sequence of which is present in SEQ ID NO: 1. Yes. A list of such sequences is provided in Table 1. In one embodiment, a preferred group of sequences present in SEQ ID NO: 1 are SEQ ID NOs: 4-15.

  Sequence motifs in the target (referred to as “hot spots”) to which these preferred oligomeric compounds are complementary are preferred sites for targeting. Motifs referred to in the sequence listing are referred to as DNA sequences-but they may refer to other nucleotides or nucleotide analogs that retain the same sequence or base subsequence in a contiguous sequence of compounds. It should be noted.

  The present invention provides oligomeric compounds comprising a total of 10-15 nucleobases, the sequence of which is present in SEQ ID NO: 1 and comprising at least 2 or at least 3 nucleotide analogs.

  Suitably, the oligomeric compound of the invention consists of a continuous nucleobase sequence of nucleobases totaling 10-15 in length.

  In one embodiment, the sequence of the nucleobases is SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 (motif sequence), or their corresponding Selected from the group consisting of subsequences.

  'Corresponding subsequence' means that the oligomer of the present invention is selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15. A situation in which a contiguous sequence of bases present in the oligomer, but shorter than the respective sequence, is found in the respective sequence.

  The terms “corresponding to” and “corresponding” refer to the reverse complement of the nucleobase sequence of the oligomer (consecutive nucleobase sequence) and i) the mRNA encoding the nucleic acid target, eg ApoB target protein. And / or ii) refers to a comparison between the nucleotide sequences provided herein, eg, the equivalent nucleotide sequences of the group consisting of SEQ ID NOs: 1-15. A nucleotide analog is directly compared to its equal or corresponding nucleotide. Thus, in one embodiment, the terms “corresponding” / “corresponding” refer to the nucleotide or nucleotide analog combined sequence of the oligomeric compounds of the present invention, or a subsequence thereof, and apolipoprotein B Refers to a comparison between an equivalent nucleotide sequence of a nucleic acid sequence (ie, a nucleic acid target).

  The terms “corresponding nucleoside / nucleotide analog” and “corresponding nucleoside / nucleotide” are intended to indicate that the nitrogen bases in the nucleoside / nucleotide analog and nucleoside / nucleotide are identical. For example, if the 2-deoxyribose unit of nucleotides is attached to adenine, the “corresponding nucleoside analog” contains a pentose unit (different from 2-deoxyribose) attached to adenine.

  The term “nucleic acid” is defined as a molecule formed by the covalent bond of two or more nucleotides. The terms “nucleic acid” and “polynucleotide” are used interchangeably herein. For example, DNA and RNA are nucleic acids.

  In one embodiment, the compound of the invention is selected from the group consisting of SEQ ID NOs: 17, 18, 19, 20, 21, 22, 23, 24 and 25.

  In one embodiment, the compounds of the invention are selected from the group consisting of SEQ ID NOs: 26, 27, 28, 29, 30, 31, 32 and 33.

  In one embodiment, the compound of the invention is selected from the group consisting of SEQ ID NOs: 34, 35, 36, 37, 38, 39 and 40.

  In one embodiment, the compounds of the invention are selected from the group consisting of SEQ ID NOs: 16, 17, 26 and 34; or 17, 26 and 34.

  In one embodiment, the compounds of the invention are selected from the group consisting of SEQ ID NOs: 41-49.

Gapmer In a preferred embodiment, the nucleobase sequence of the compounds of the invention is in the 5 ′ to 3 ′ direction, i) region A: a series of 2-4 nucleotide analogues, ii) the following region B: a series 6-11 nucleotides (eg, DNA nucleotides), iii) followed by region C: a series of 2-4 nucleotide analogs, and optionally iv) one or two nucleotides (D) Become.

  In one embodiment, region A has a length of 1 nucleotide analog. In one embodiment, region A has a length of 2 nucleotide analogs. In one embodiment, region A has a length of 3 nucleotide analogs. In one embodiment, region A has a length of 4 nucleotide analogs. In one embodiment, region C has a length of 1 nucleotide analog. In one embodiment, region C has a length of 2 nucleotide analogs. In one embodiment, region C has a length of 3 nucleotide analogs. In one embodiment, region C has a length of 4 nucleotide analogs. In one embodiment, region B has a length between 7 and 10 nucleotides (eg, DNA nucleotides), such as 8 or 9 nucleotides (eg, DNA nucleotides). In one embodiment, the compounds of the invention have a length of 12 to 15 nucleobases. In one embodiment, the compounds of the invention have a length of 12, 13 or 14 nucleobases. In one embodiment, the gapmer can be of the formula D-A-B-C, in the 5 ′ to 3 ′ direction.

In one embodiment, the nucleobase sequence of the compound of the invention comprises a group of internucleobase linkages selected from the group consisting of a phosphate group, a phosphodiester group, a phosphorothioate group, and a boranophosphate group, and an internucleoside bond. The bond can be —OP (O) ₂ —O—, —OP (O, S) —O—. In one embodiment, the internucleoside linkage is a phosphate group and / or a phosphorothioate group. In certain embodiments, every nucleotide comprises a phosphorothioate group. In one embodiment, some or all of the nucleotides are linked to each other by a phosphorothioate group. Suitably all nucleotides are linked to each other by a phosphorothioate group.

  In one embodiment, the group of internucleobase linkages between nucleobase units of the nucleobase sequence of the compounds of the invention is independently selected from either phosphorothioate or phosphodiester linkage groups.

  In one embodiment, region A comprises at least one phosphodiester bond between two nucleotide analog units or between a nucleotide analog unit and a nucleotide unit. In this context, it will be understood that the linking group between the nucleotide analog and the nucleotide unit refers to the linking group between regions A and B.

  In one embodiment, region C comprises at least one phosphodiester bond between two nucleotide analog units or between a nucleotide analog unit and a nucleotide unit. In this context, it will be understood that the linking group between the nucleotide analog and the nucleotide unit refers to the linking group between regions B and C.

  In one embodiment, the internucleotide linkage between the nucleotides of region B is phosphorothioate. In one embodiment, the internucleobase linkage between the 3 ′ nucleotide analog of A and the 5 ′ nucleotide of region B is phosphorothioate.

  In one embodiment, the internucleobase linkage between the 3 ′ nucleotide of region B and the 5 ′ nucleotide analog of region C is phosphorothioate. In one embodiment, the internucleobase linkage between the 3 ′ nucleotide analog of A and the 5 ′ nucleotide of region B is a phosphodiester. In one embodiment, the internucleobase linkage between the 3 ′ nucleotide of region B and the 5 ′ nucleotide analog of region C is a phosphodiester. In one embodiment, the internucleobase linkage between the two 5 ′ nucleotide analogs of region A is a phosphodiester. In one embodiment, the internucleobase linkage between the two 3 ′ nucleotide analogs of region C is a phosphodiester. In one embodiment, the internucleobase linkage between the two 3 ′ nucleotide analogs of region A is a phosphodiester. In one embodiment, the internucleobase linkage between the two 5 ′ nucleotide analogs of region C is a phosphodiester. In one embodiment, region A has a length of 4 nucleotide analogs and the internucleobase linkage between the central two nucleotide analogs of region A is a phosphodiester. In one embodiment, region C has a length of 4 nucleotide analogs and the internucleobase linkage between the central two nucleotide analogs of region C is a phosphodiester. In one embodiment, all internucleobase linkages between nucleotide analogs present in the compounds of the invention are phosphodiesters.

  In one embodiment, eg, the above embodiment that is referred to as being appropriate and not specifically shown, all remaining internucleobase linkages are either phosphodiesters or phosphorothioates, or another embodiment Is a mixture of them. In one embodiment, all internucleobase linkage groups are phosphorothioate.

ホスホロチオエート(phosphorthioate)

2'-O-メチル

2'-MOE

2'-フルオロ

2'-AP

HNA

CeNA

PNA

モルホリノ

2'-F-ANA

2'-(3-ヒドロキシ)プロピル

3'-ホスホロアミダート

ボラノホスフェート

模式図 1 Nucleotide analogs As used herein, the term “nucleotide analog” refers to unnatural nucleotides, for example, in one preferred embodiment, the ribose unit is different from 2-deoxyribose and / or the nitrogen base is A, Unlike C, T and G and / or different phosphate linkage groups between nucleosides. Specific examples of nucleoside analogs are described, for example, by Freier &Altmann; Nucl. Acid Res., 1997, 25, 4429-4443 and Uhlmann; Curr. Opinion in Drug Development, 2000, 3 (2), 293-213, As well as in schematic diagram 1:

Phosphorothioate

2'-O-methyl

2'-MOE

2'-Fluoro

2'-AP

HNA

CeNA

PNA

Morpholino

2'-F-ANA

2 '-(3-hydroxy) propyl

3'-phosphoramidates

Boranophosphate

Schematic 1

  Suitable nucleotide analogs for use in the oligonucleotides of the invention are independently selected from the group consisting of: 2'-O-alkyl-RNA monomers, 2'-amino-DNA monomers, 2'-fluoro -DNA monomers, LNA monomers, arabino nucleic acid (ANA) monomers, 2'-fluoro-ANA monomers, HNA monomers, INA monomers.

  2'-O-methoxyethyl-RNA, 2'-fluoro-DNA monomers and LNA are preferred, as such, the oligonucleotides of the present invention are nucleotide analogues independently selected from these three types of analogues Or may consist of only one type selected from the three types. In the most preferred embodiment, the oligonucleotide comprises only LNA nucleotide analogs and nucleotides (RNA or DNA, most preferably DNA nucleotides).

  In one embodiment, nucleotide analogs present in the compounds of the invention, eg, in regions A and C, are independently selected from the group consisting of: 2′-O-alkyl-RNA monomers, 2′-amino -DNA monomers, 2'-fluoro-DNA monomers, LNA monomers, arabino nucleic acid (ANA) monomers, 2'-fluoro-ANA monomers, HNA monomers, INA monomers.

  In one embodiment, the nucleotide analog is independently selected from the group consisting of 2'-MOE-RNA (2'-O-methoxyethyl-RNA), 2'fluoro-DNA, and LNA.

  In one embodiment, at least one of the nucleotide analogs is a locked nucleic acid (LNA).

  In one embodiment, at least one of said nucleotide analogs is 2'-MOE-RNA, for example 2, 3, 4, 5, 6, 7 or 8 2'-MOE-RNA nucleobase units.

  In one embodiment, at least one of said nucleotide analogs is 2'-fluoro DNA, such as 2, 3, 4, 5, 6, 7 or 8 2'-fluoro-DNA nucleobase units.

  In one embodiment, 2, 3, 4, 5, 6, 7 or 8 of the nucleotide analogs are LNA, and any remaining nucleotide analogs are selected from the group of nucleotide analogs referred to herein. Can be done.

  The oligomeric compounds of the invention preferably comprise at least 2 or at least 3 nucleotide analogues. At least 2 or at least 3 nucleotide analogues are preferably locked nucleic acid nucleotide analogues (LNA), and oligomeric compounds comprising such nucleotide analogues are referred to herein as “LNA oligomeric compounds”, “LNA oligonucleotides”. Referred to as “compounds” and “LNA oligonucleotides”.

  In one embodiment, all nucleotide analogs are LNA.

  In one embodiment, all nucleotide analogs are not PNA.

  In one embodiment, the LNA nucleotide analogs present in the compounds of the invention are beta-D-oxy-LNA, alpha-L-oxy-LNA, beta-D-amino-LNA and beta-D-thio-LNA. Selected from.

  Preferably, the oligomeric compound, such as an antisense oligonucleotide, can comprise both LNA and DNA units. Preferably, the combined total of LNA and DNA units is between 10-15 including 11, 12, 13 and 14.

  In one embodiment, all LNA C residues in the oligomeric compounds of the invention comprising LNA, such as antisense oligonucleotides, are 5 ′ methyl-cytosines.

[式中、X は -O-、-S-、-N(R^N*)-、-C(R⁶R^6*)- から選択される;
B は、水素、所望により置換された C_1-4-アルコキシ、所望により置換された C_1-4-アルキル、所望により置換された C_1-4-アシルオキシ、天然または非天然の核酸塩基、光化学的に活性な基、熱化学的に活性な基、キレート基(chelating group)、レポーター基、およびリガンド(ligand)から選択される;
P は、後に続くモノマーまたは 5'-末端基とのヌクレオシド間結合のためのラジカルの位置を示し、かかるヌクレオシド間結合または 5'-末端基は所望により、置換基 R⁵ または等しく適用できる(equally applicable)置換基 R^5* を含む;
P* は、先行するモノマーまたは 3'-末端基とのヌクレオシド間結合を示す;
R^4* および R^2* は共に、-C(R^aR^b)-、-C(R^a)=C(R^b)-、-C(R^a)=N-、-O-、-Si(R^a)₂-、-S-、-SO₂-、-N(R^a)-、および >C=Z から選択される 1-4 の基/原子からなるビラジカルを示し、
ここで、Z は O、S、および N(R^a)から選択され、
R^a および R^b は各々独立して水素、所望により置換された C_1-12-アルキル、所望により置換された C_2-12-アルケニル、所望により置換された C_2-12-アルキニル、ヒドロキシ、C_1-12-アルコキシ、C_2-12-アルコキシアルキル、C_2-12-アルケニルオキシ、カルボキシ、C_1-12-アルコキシカルボニル、C_1-12-アルキルカルボニル、ホルミル、アリール、アリールオキシ-カルボニル、アリールオキシ、アリールカルボニル、ヘテロアリール、ヘテロアリールオキシ-カルボニル、ヘテロアリールオキシ、ヘテロアリールカルボニル、シクロアルキル、シクロアルキルオキシ-カルボニル、シクロアルキルオキシ、シクロアルキルカルボニル、ヘテロシクロアルキル、ヘテロシクロアルキルオキシ-カルボニル、ヘテロシクロアルキルオキシ、ヘテロシクロアルキルカルボニル、アミノ、モノおよびジ(C_1-6-アルキル)アミノ、トリス(C_1-6-アルキル)アンモニウム、カルバモイル、モノおよびジ(C_1-6-アルキル)-アミノ-カルボニル、アミノ-C_1-6-アルキル-アミノカルボニル、モノおよびジ(C_1-6-アルキル)アミノ-C_1-6-アルキル-アミノカルボニル、C_1-6-アルキル-カルボニルアミノ、カルバミド(carbamido)、C_1-6-アルカノイルオキシ、スルホノ、C_1-6-アルキルスルホニルオキシ、ニトロ、アジド、スルファニル、C_1-6-アルキルチオ、ハロゲン、DNA インターカレーター、光化学的に活性な基、熱化学的に活性な基、キレート基、レポーター基およびリガンドから選択され、ここで、アリールおよびヘテロアリールは所望により置換されてもよく、２つのジェミナルな置換基 R^a および R^b は共に、所望により置換されたメチレン(=CH₂)を示し得るか、または共に所望により -O-、-S- および -(NR^N)- から選択される１以上のヘテロ原子/基によって分断され(interrupted)および/または終結させられる(terminated) 1-5 炭素原子のアルキレン鎖からなるスピロ ビラジカルを形成し得、ここで R^N は水素および C_1-4-アルキルから選択され、そして、
置換基 R^1*、R²、R³、R⁵、R^5*、R⁶ および R^6* の各々は、水素、所望により置換された C_1-12-アルキル、所望により置換された C_2-12-アルケニル、所望により置換された C_2-12-アルキニル、ヒドロキシ、C_1-12-アルコキシ、C_2-12-アルコキシアルキル、C_2-12-アルケニルオキシ、カルボキシ、C_1-12-アルコキシカルボニル、C_1-12-アルキルカルボニル、ホルミル、アリール、アリールオキシ-カルボニル、アリールオキシ、アリールカルボニル、ヘテロアリール、ヘテロアリールオキシ-カルボニル、ヘテロアリールオキシ、ヘテロアリールカルボニル、シクロアルキル、シクロアルキルオキシ-カルボニル、シクロアルキルオキシ、シクロアルキルカルボニル、ヘテロシクロアルキル、ヘテロシクロアルキルオキシ-カルボニル、ヘテロシクロアルキルオキシ、ヘテロシクロアルキルカルボニル、アミノ、モノおよびジ(C_1-6-アルキル)アミノ、カルバモイル、モノおよびジ(C_1-6-アルキル)-アミノ-カルボニル、アミノ-C_1-6-アルキル-アミノカルボニル、モノおよびジ(C_1-6-アルキル)アミノ-C_1-6-アルキル-アミノカルボニル、C_1-6-アルキル-カルボニルアミノ、カルバミド(carbamido)、C_1-6-アルカノイルオキシ、スルホノ、C_1-6-アルキルスルホニルオキシ、ニトロ、アジド、スルファニル、C_1-6-アルキルチオ、ハロゲン、DNA インターカレーター、光化学的に活性な基、熱化学的に活性な基、キレート基、レポーター基およびリガンドから独立に選択され、ここで、アリールおよびヘテロアリールは所望により置換されてもよく、２つのジェミナルな置換基は共に、オキソ、チオキソ、イミノまたは所望により置換されたメチレンを示し得るか、または共に所望により -O-、-S- および -(NR^N)- から選択される１以上のヘテロ原子/基によって分断されおよび/または終結させられる 1-5 炭素原子のアルキレン鎖からなるスピロ ビラジカルを形成し得、ここで、R^N は水素および C_1-4-アルキルから選択され、２つの隣接した(ジェミナルでない)置換基は二重結合をもたらすさらなる結合を示し得; R^N* は、それが存在し、かつビラジカルに含まれない場合には、水素および C_1-4-アルキルから選択される;] Locked nucleic acid (LNA)
The term “LNA” refers to a bicyclic nucleotide analog known as “locked nucleic acid”. The term can refer to an LNA monomer or, when used in the context of an “LNA oligonucleotide”, refers to an oligonucleotide containing one or more such bicyclic nucleotide analogs. The LNA used in the oligonucleotide compounds of the present invention preferably has the structure of general formula I and its basic and acid addition salts:

Wherein X is selected from -O-, -S-, -N (R ^{N *} )-, -C (R ⁶ R ^{6 *} )-;
B is hydrogen, optionally substituted C _1-4 -alkoxy, optionally substituted C _1-4 -alkyl, optionally substituted C _1-4 -acyloxy, natural or non-natural nucleobase, photochemistry Active groups, thermochemically active groups, chelating groups, reporter groups, and ligands;
P indicates the position of the radical for subsequent internucleoside linkage with the monomer or 5′-end group, such internucleoside linkage or 5′-end group being optionally applicable to the substituent R ⁵ or equivalently (equally applicable) containing the substituent R ^{5 *} ;
P * indicates an internucleoside linkage with the preceding monomer or 3'-end group;
R ^{4 *} and R ^{2 *} are both -C (R ^a R ^b )-, -C (R ^a ) = C (R ^b )-, -C (R ^a ) = N-, -O-, -Si (R ^a ) _2- , -S-, -SO _2- , -N (R ^a )-, and a biradical consisting of 1-4 groups / atoms selected from> C = Z
Where Z is selected from O, S, and N (R ^a )
R ^a and R ^b are each independently hydrogen, optionally substituted C _1-12 -alkyl, optionally substituted C _2-12 -alkenyl, optionally substituted C _2-12 -alkynyl, hydroxy, C _1-12 -alkoxy, C _{2-12 -alkoxyalkyl} , C _2-12 -alkenyloxy, carboxy, C _1-12 -alkoxycarbonyl, C _1-12 -alkylcarbonyl, formyl, aryl, aryloxy-carbonyl, Aryloxy, arylcarbonyl, heteroaryl, heteroaryloxy-carbonyl, heteroaryloxy, heteroarylcarbonyl, cycloalkyl, cycloalkyloxy-carbonyl, cycloalkyloxy, cycloalkylcarbonyl, heterocycloalkyl, heterocycloalkyloxy-carbonyl , Heterocycloalkyloxy, heterocycloal Le carbonyl, amino, mono- and di (C _1-6 - alkyl) amino, tris (C _1-6 - alkyl) ammonium, carbamoyl, mono- and di (C _1-6 - alkyl) - amino - carbonyl, amino -C _1-6 -alkyl-aminocarbonyl, mono and di (C _1-6 -alkyl) amino-C _1-6 -alkyl-aminocarbonyl, C _1-6 -alkyl-carbonylamino, carbamido, C _{1- 6} -alkanoyloxy, sulfono, C _1-6 -alkylsulfonyloxy, nitro, azide, sulfanyl, C _1-6 -alkylthio, halogen, DNA intercalator, photochemically active group, thermochemically active group, is selected from the chelating group, reporter groups, and ligands, substituted wherein aryl and heteroaryl may be optionally substituted, two geminal substituents R ^a and R ^b are both optionally Methylene (= CH ₂₎ or may show, or together optionally -O -, - S- and - (NR ^N) - interrupted by one or more heteroatoms / groups selected from (interrupted) and / Or a terminated spirobiradical consisting of an alkylene chain of 1-5 carbon atoms, where R ^N is selected from hydrogen and C _1-4 -alkyl, and
Each of the substituents R ^{1 *} , R ² , R ³ , R ⁵ , R ^{5 *} , R ⁶ and R ^{6 *} is hydrogen, optionally substituted C _1-12 -alkyl, optionally substituted C _{2 -12} -alkenyl, optionally substituted C _2-12 -alkynyl, hydroxy, C _1-12 -alkoxy, C _{2-12 -alkoxyalkyl} , C _2-12 -alkenyloxy, carboxy, C _1-12 -alkoxy Carbonyl, C _1-12 -alkylcarbonyl, formyl, aryl, aryloxy-carbonyl, aryloxy, arylcarbonyl, heteroaryl, heteroaryloxy-carbonyl, heteroaryloxy, heteroarylcarbonyl, cycloalkyl, cycloalkyloxy-carbonyl , Cycloalkyloxy, cycloalkylcarbonyl, heterocycloalkyl, heterocycloalkyloxy-carbonyl, heterocycloal Yloxy, Heterocycloalkylcarbonyl, amino, mono- and di (C _1-6 - alkyl) amino, carbamoyl, mono- and di (C _1-6 - alkyl) - amino - carbonyl, amino -C _1-6 - alkyl - amino Carbonyl, mono and di (C _1-6 -alkyl) amino-C _1-6 -alkyl-aminocarbonyl, C _1-6 -alkyl-carbonylamino, carbamido, C _1-6 -alkanoyloxy, sulfono, C _1-6 -alkylsulfonyloxy, nitro, azide, sulfanyl, C _1-6 -alkylthio, halogen, DNA intercalator, photochemically active group, thermochemically active group, chelating group, reporter group and ligand Wherein aryl and heteroaryl may be optionally substituted, and the two geminal substituents may both be oxo, thioxo, imino or Whether may represents a methylene which is optionally substituted, or together optionally -O -, - brought interrupted by one or more heteroatoms / groups selected from and / or terminate - S- and - (NR ^N) 1-5 can form a spiro biradical consisting of alkylene chain of carbon atoms, wherein, R ^N is hydrogen and C _1-4 - is selected from alkyl, two were adjacent (not geminal) substituents a double bond May show additional bonds that result; R ^{N *} is selected from hydrogen and C _1-4 -alkyl, if it is present and not included in the biradical;

In one embodiment, R ^{5 *} is selected from H, —CH ₃ , —CH ₂ —CH ₃ , —CH ₂ —O—CH ₃ and —CH═CH ₂ .

In one embodiment, R ^{4 *} and R ^{2 *} are both -C (R ^a R ^b ) -O-, -C (R ^a R ^b ) -C (R ^c R ^d ) -O-, -C ( R ^a R ^b ) -C (R ^c R ^d ) -C (R ^e R ^f ) -O-, -C (R ^a R ^b ) -OC (R ^c R ^d )-, -C (R ^a R ^b ) -OC (R ^c R ^d ) -O-, -C (R ^a R ^b ) -C (R ^c R ^d )-, -C (R ^a R ^b ) -C (R ^c R ^d ) -C ( R ^e R ^f )-, -C (R ^a ) = C (R ^b ) -C (R ^c R ^d )-, -C (R ^a R ^b ) -N (R ^c )-, -C (R ^a R ^b ) -C (R ^c R ^d ) -N (R ^e )-, -C (R ^a R ^b ) -N (R ^c ) -O-, and -C (R ^a R ^b ) -S-, -C (R ^a R ^b ) -C (R ^c R ^d ) -S-, wherein R ^a , R ^b , R ^c , R ^d , R ^e and R ^f are each Hydrogen, optionally substituted C _1-12 -alkyl, optionally substituted C _2-12 -alkenyl, optionally substituted C _2-12 -alkynyl, hydroxy, C _1-12 -alkoxy, C _{2- 12} - alkoxyalkyl, C _2-12 - alkenyloxy, carboxy, C _1-12 - alkoxycarbonyl, C _1-12 - alkyl, formyl, aryl, aryl Oxy-carbonyl, aryloxy, arylcarbonyl, heteroaryl, heteroaryloxy-carbonyl, heteroaryloxy, heteroarylcarbonyl, cycloalkyl, cycloalkyloxy-carbonyl, cycloalkyloxy, cycloalkylcarbonyl, heterocycloalkyl, heterocyclo Alkyloxy-carbonyl, heterocycloalkyloxy, heterocycloalkylcarbonyl, amino, mono and di (C _1-6 -alkyl) amino, carbamoyl, mono and di (C _1-6 -alkyl) -amino-carbonyl, amino- C _1-6 -alkyl-aminocarbonyl, mono and di (C _1-6 -alkyl) amino-C _1-6 -alkyl-aminocarbonyl, C _1-6 -alkyl-carbonylamino, carbamido, C _{1 -6} -alkanoyloxy, sulfono, C _1-6 -al Independently selected from killsulfonyloxy, nitro, azide, sulfanyl, C _1-6 -alkylthio, halogen, DNA intercalator, photochemically active group, thermochemically active group, chelating group, reporter group and ligand Where aryl and heteroaryl may be optionally substituted, and the two geminal substituents may both represent optionally substituted methylene (= CH ₂ ), or both optionally -O-, A spirobiradical consisting of an alkylene chain of 1-5 carbon atoms interrupted and / or terminated by one or more heteroatoms / groups selected from -S-, and-(NR ^N )-, where , R ^N is selected from hydrogen and C _1-4 -alkyl.

In a further embodiment, R ^{4 *} and R ^{2 *} are _{both, -CH 2 -O -, - CH} 2 -S -, - CH 2 -NH -, - CH 2 -N (CH 3) -, - CH 2 - _{CH 2 -O -, - CH 2} -CH (CH 3) -, - CH 2 -CH 2 -S -, - CH 2 -CH 2 -NH -, - CH 2 -CH 2 -CH 2 -, - CH ₂ -CH ₂ -CH ₂ -O-, -CH ₂ -CH ₂ -CH (CH ₃ )-, -CH = CH-CH _2- , -CH ₂ -O-CH ₂ -O-, -CH _{2- NH-O -, - CH 2} -N (CH 3) -O -, - CH 2 -O-CH 2 -, - CH (CH 3) -O -, - CH (CH 2 -O-CH 3) - Indicates a biradical selected from O-.

  All chiral centers can be found in either R or S orientation.

[式中、Y は -O-、-S-、-NH- または N(R^H)である; Z および Z* は独立に、ヌクレオシド間結合、末端基または保護基から選択される; B は、天然または非天然のヌクレオチド塩基部分を構成し、R^H は、水素および C_1-4-アルキルから選択される。] Preferably, the LNA used in the oligomer of the invention comprises at least one LNA unit of any of the following formulas:

Wherein Y is -O-, -S-, -NH- or N (R ^H ); Z and Z * are independently selected from internucleoside linkages, end groups or protecting groups; B is Constituting a natural or non-natural nucleotide base moiety, R ^H is selected from hydrogen and C _1-4 -alkyl. ]

  The term “thio-LNA” includes locked nucleotides where Y in the above formula represents S. Thio-LNA can be in both beta-D and alpha-L configurations.

The term “amino-LNA” refers to a lock wherein Y is selected from —N (H) — and N (R ^H ) —, wherein R ^H is selected from hydrogen and C _1-4 -alkyl. Containing nucleotides. Amino-LNA can be in both beta-D and alpha-L configurations.

  The term “oxy-LNA” includes locked nucleotides where Y in the above formula represents —O—. Oxy-LNA can be in both beta-D and alpha-L configurations.

The term “ENA” is locked when Y in the above formula is —CH ₂ —O— (where the oxygen atom of —CH ₂ —O— is bonded 2 ′ to the base B) Contains nucleotides. ENA can be in both beta-D and alpha-L configurations.

  In a preferred embodiment, the LNA is selected from beta-D-oxy-LNA, alpha-L-oxy-LNA, beta-D-amino-LNA and beta-D-thio-LNA, particularly in beta-D-oxy-LNA is there.

β-D-オキシ-LNA

α-L-オキシ-LNA

β-D-チオ-LNA

β-D-ENA

β-D-アミノ-LNA

模式図 2 A particularly preferred LNA unit is shown in schematic diagram 2:

β-D-oxy-LNA

α-L-oxy-LNA

β-D-thio-LNA

β-D-ENA

β-D-amino-LNA

Schematic diagram 2

X および Y は、-O-、-S-、-N(H)-、N(R)-、-CH₂- もしくは -CH- (二重結合の一部である場合)、-CH₂-O-、-CH₂-S-、-CH₂-N(H)-、-CH₂-N(R)-、-CH₂-CH₂- もしくは -CH₂-CH- (二重結合の一部である場合)、-CH=CH- の基から独立に選択され、ここで R は水素および C_1-4-アルキルから選択される; Z および Z^* は、ヌクレオシド間結合、末端基または保護基から独立に選択される; B は、天然または非天然の核酸塩基を構成する; そして不斉基は、いずれかの配向において見出され得る。 In one embodiment, the locked nucleic acid (LNA) used in the oligonucleotide compounds of the invention has a structure of the general formula:

X and Y are -O-, -S-, -N (H)-, N (R)-, -CH ₂ -or -CH- (if part of a double bond), -CH _{2- O -, - CH 2 -S -} , - CH 2 -N (H) -, - CH 2 -N (R) -, - CH 2 -CH 2 - or -CH ₂ -CH- (double binding single And R is selected from hydrogen and C _1-4 -alkyl; Z and Z ^* are internucleoside linkages, end groups or protections. Selected independently from the group; B constitutes a natural or non-natural nucleobase; and asymmetric groups can be found in either orientation.

  The components of LNA nucleotide analogs (e.g., β-D-oxy-LNA, β-D-thio-LNA, β-D-amino-LNA and α-L-oxy-LNA) are in accordance with established and published procedures. Can be prepared-see, for example, WO2007 / 031081, which is incorporated herein by reference.

Preferably, LNA used in the oligomer of the present invention is -OP (O) ₂ -O-, -OP (O, S) -O-, -OP (S) ₂ -O-, -SP (O) ₂ -O-, -SP (O, S) -O-, -SP (S) ₂ -O-, -OP (O) ₂ -S-, -OP (O, S) -S-, -SP (O ) ₂ -S-, -O-PO (R ^H ) -O-, O-PO (OCH ₃ ) -O-, -O-PO (NR ^H ) -O-, -O-PO (OCH ₂ CH ₂ SR) -O-, -O-PO (BH ₃ ) -O-, -O-PO (NHR ^H ) -O-, -OP (O) ₂ -NR ^H- , -NR ^H -P (O) ₂ Including an internucleoside linkage selected from —O—, —NR ^H —CO—O—, wherein R ^H is selected from hydrogen and C _1-4 -alkyl.

  As used herein, the term “DNA intercalator” means a group capable of intervening between a DNA or RNA helix, duplex or triplex. (Examples of functional parts of DNA intercalators are acridine, anthracene, quinones such as anthraquinone, indole, quinoline, isoquinoline, dihydroquinone, anthracycline, tetracycline, methylene blue, anthracyclinone, psoralen, coumarin, ethidium halide. Dynemicin, metal complexes, such as 1,10-phenanthroline-copper, tris (4,7-diphenyl-1,10-phenanthroline) ruthenium-cobalt-enediyne, such as calcheamicin, porphyrin, distamycin Netropcin, viologen, daunomycin.) Particularly interesting examples are acridine, quinones such as anthraquinone, methylene blue, psoralen, coumarin, and ethidium halide.

  As used herein, the term “photochemically active group” includes a compound that can undergo a chemical reaction when irradiated with light. Specific examples of this functional group are quinones, especially 6-methyl-1,4-naphthoquinone, anthraquinone, naphthoquinone and 1,4-dimethyl-anthraquinone, diazirine, aromatic azide, benzophenone, psoralen, diazo compounds, As well as diazirino compounds.

  As used herein, a “thermochemically reactive group” is defined as a functional group that can undergo thermochemically induced covalent bond formation with other groups. Specific examples of functional moieties of thermochemically reactive groups include carboxylic acids, carboxylic esters, such as activated esters, carboxylic halides, such as acid fluoride, acid chloride, acid bromide and acid iodide, carboxylic acid azide, Carboxylic acid hydrazide, sulfonic acid, sulfonic acid ester, sulfonic acid halide, semicarbazide, thiosemicarbazide, aldehyde, ketone, primary alcohol, secondary alcohol, tertiary alcohol, phenol, alkyl halide, thiol, disulfide, primary amine, secondary amine Tertiary amines, hydrazines, epoxides, maleimides and boronic acid derivatives.

  As used herein, the term “chelating group” means a molecule that contains more than one binding site and frequently binds to another molecule, atom or ion through more than one binding site at the same time. Examples of functional moieties of chelating groups are iminodiacetic acid, nitrilotriacetic acid, ethylenediaminetetraacetic acid (EDTA), aminophosphonic acid, and the like.

As used herein, the term “reporter group” means a group that is detectable by itself or as part of a detection series. Examples of functional parts of reporter groups are biotin, digoxigenin, fluorescent groups (electromagnetic radiation, e.g. light or X-rays of a certain wavelength can be absorbed, and then the absorbed energy is Specific examples include dansyl (5-dimethylamino) -1-naphthalenesulfonyl), doxyl (DOXYL) (N-oxyl-4,4-dimethyloxazolidine), proxyl (PROXYL) (N-oxyl). -2,2,5,5-tetramethylpyrrolidine), TEMPO (N-oxyl-2,2,6,6-tetramethylpiperidine), dinitrophenyl, acridine, coumarin, Cy3 and Cy5 (Biological Detection Systems , Inc., erytrosine, coumaric acid, umbelliferone, Texas Red, rhodamine, tetramethylrhodamine, Rox, 7-nitrobenzo-2-oxa-1-diazole (NBD), pyrene , Fluor In, europium, ruthenium, samarium, and other rare earth metals), radioisotope labels, chemiluminescent labels (labels detectable through the emission of light during chemical reactions), spin labels (to biological molecules) Bound free radicals detectable by use of electron spin resonance spectroscopy (e.g. substituted organic nitroxides) or other paramagnetic probes (e.g. Cu2 ⁺ , Mg2 ⁺ )), enzymes (e.g. peroxidase, alkaline phosphatase, β-galactosidase, and glucose oxidase), antigens, antibodies, haptens (groups that can bind to antibodies but cannot elicit an immune response by themselves, such as peptides and steroid hormones), such as Transport systems for cell membrane penetration: fatty acid residues, steroid moieties (cholesterol), vitamin A, Vitamin D, vitamin E, folic acid, peptides for specific receptors, groups that mediate food and drink, epidermal growth factor (EGF), bradykinin, and platelet-derived growth factor (PDGF). Particularly interesting examples are biotin, fluorescein, Texas Red, rhodamine, dinitrophenyl, digoxigenin, ruthenium, europium, Cy5 and Cy3.

As used herein, “ligand” means a substance that binds. The ligand may be, for example, the following functional groups: aromatic groups (e.g. benzene, pyridine, naphthalene, anthracene, and phenanthrene), heteroaromatic groups (e.g. thiophene, furan, tetrahydrofuran, pyridine, dioxane, and pyrimidine), carvone Acid, carboxylic acid ester, carboxylic acid halide, carboxylic acid azide, carboxylic acid hydrazide, sulfonic acid, sulfonic acid ester, sulfonic acid halide, semicarbazide, thiosemicarbazide, aldehyde, ketone, primary alcohol, secondary alcohol, tertiary alcohol, phenol , Alkyl halides, thiols, disulfides, primary amines, secondary amines, tertiary amines, hydrazines, epoxides, maleimides, optionally one or more heteroatoms such as oxygen atoms, nitrogen atoms, and / or Obtained include C ₁ -C ₂₀ alkyl group which is allowed to or terminated interrupted by a sulfur atom, optionally aromatic or monounsaturated / polyunsaturated hydrocarbons, polyoxyethylene such as polyethylene glycol, oligo / polyamides Including, for example, poly-β-alanine, polyglycine, polylysine, peptides, oligosaccharides / polysaccharides, oligo / polyphosphates, toxins, antibiotics, cytotoxins and steroids, and “affinity ligands” ie specific proteins, antibodies, Functional groups or biomolecules having specific affinity for sites on polysaccharides and oligosaccharides and other biomolecules may also be included.

Complexes The present invention also provides complexes comprising a compound of the invention and at least one non-nucleotide or non-polynucleotide moiety covalently linked to the compound. PCT / DK2006 / 000512, which is incorporated herein by reference, provides suitable ligands and complexes.

  In one embodiment of the invention, the oligonucleotide may bind to a ligand / complex that can be used, for example, to increase the uptake of the oligonucleotide into the cell. This binding can occur at the terminal position 5 '/ 3'-OH, but the ligand can also occur at sugars and / or bases. 3'-OH is a preferred site for cholesterol binding.

  In preferred embodiments, the oligonucleotides of the invention bind to moieties that improve uptake in vivo, such as cholesterol.

  Thus, an oligomeric compound can be, for example, a peptide nucleic acid (PNA), a protein (eg, an antibody against a target protein), a macromolecule, a low molecular weight drug substance, such that the oligomeric compound can be arranged in a dimeric or dendritic structure. (drug substance), fatty acid chain, sugar residue, glycoprotein, polymer (eg polyethylene glycol), micelle forming group, antibody, carbohydrate, receptor binding group, steroid, eg cholesterol, polypeptide, intercalate It can be combined with non-nucleotide or non-polynucleotide moieties including agents such as acridine derivatives, long chain alcohols, dendrimers, phospholipids and other lipophilic groups or combinations thereof, or can form a chimera.

  In one embodiment relating to the complex, the non-nucleotide or non-polynucleotide moiety consists of or comprises a sterol group, such as cholesterol.

  Other such non-nucleotide or non-polynucleotide moieties include, but are not limited to, lipid moieties such as cholesterol moieties, cholic acid, thioethers such as hexyl-s-trityl thiol, thiocholesterol, aliphatic chains such as dodecandiol. ) Or undecyl residues, phospholipids such as di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-o-hexadecyl-rac-glycero-3-h-phosphonate, polyamine or polyethylene glycol chains, adamantaneacetic acid, palmityl Containing a moiety, octadecylamine or hexylamino-carbonyl-oxycholesterol moiety.

  The oligomers of the invention may also bind to active drug substances such as aspirin, ibuprofen, sulfa drugs, antidiabetics, antibacterials or antibiotics.

Pharmaceutical Composition The present invention further provides a pharmaceutical composition comprising a compound of the present invention or a complex of the present invention and a pharmaceutically acceptable diluent, carrier or adjuvant.

  Pharmaceutical and other compositions comprising the oligonucleotide compounds of the present invention are provided by the present invention.

  The pharmaceutical composition may further comprise, in one embodiment, at least one compound that lowers cholesterol.

  Suitable cholesterol lowering compounds include bile salt scavenging resins (e.g. cholestyramine, colestipol, and colesevelam hydrochloride), HMGCoA-reductase inhibitors (e.g. lovastatin, cerivastatin, prevastatin, atorvastatin, simvastatin, and fluvastatin) , Nicotinic acid, fibric acid derivatives (e.g. clofibrate, gemfibrozil, fenofibrate, bezafibrate, and ciprofibrate), probucol, neomycin, dextrothyroxine, plant stanol esters, cholesterol absorption inhibitors (e.g. ezetimibe), imputapide, bile acids Inhibitor of transporter (apical sodium-dependent bile acid transporter), regulator of liver CYP7a, estrogen Charging therapy (e.g., tamoxifen), and anti-inflammatory agents (e.g., glucocorticoids) may be selected from the group consisting of. A combination with statins is particularly preferred.

  Examples of statins include atorvastatin ™, cerivastatin ™, fluvastatin ™, lovastatin ™, mevastatin ™, pitavastatin ™, pravastatin ™, rosuvastatin ™, and simvastatin ™.

  The combination of a compound of the present invention and a statin may allow for a reduction in statin dosage, resulting in, for example, myalgia, muscle cramps, gastrointestinal symptoms, liver enzyme derangement, myositis, myopathy, muscle breakdown products Can overcome the side effects associated with normal doses of statins, including rhabdomyolysis (pathological disruption of skeletal muscle), which can cause acute renal failure when damaging the kidneys.

  Fibrate, a class of amphiphilic carboxylic acids, is an alternative to a compound often combined with the use of statins, despite the increased frequency of rhabdomyolysis reported for the combination of statins and fibrates. Class. Therefore, the composition of the present invention may further contain a fibrrate and optionally a statin.

  In another embodiment, the composition of the present invention contains one or more antisense compounds that target a first nucleic acid, in particular an oligonucleotide, and one or more additional antisense compounds that target a second nucleic acid target. Can do.

  In the method of the present invention, two or more combined compounds may be used together or sequentially.

  The present invention also includes oligomeric compounds of the present invention and additional compounds capable of modulating serum cholesterol levels, such as antisense oligonucleotides (oligomers) that target the nucleic acid target of PCSK9 modulators, particularly PCSK9--for example, by reference. Pharmaceutical compositions comprising those disclosed in PCT / EP2007 / 060703 which are incorporated in the specification are provided.

  The present invention also includes oligomeric compounds of the present invention and additional compounds capable of modulating serum cholesterol levels, such as antisense oligonucleotides (oligomers) that target nucleic acid targets of FABP4 modulators, particularly FABP4—for example, by reference. Pharmaceutical compositions are provided comprising those disclosed in US Provisional Application No. 60 / 969,016, incorporated herein.

Applications There is further provided a method of modulating the expression of apolipoprotein B in a cell or tissue comprising contacting the cell or tissue with one or more oligonucleotide compounds or compositions of the invention. Animals having or suspected of having a disease or condition associated with the expression of apolipoprotein B by administering a therapeutically or prophylactically effective amount of one or more oligonucleotide compounds or compositions of the invention Or a method of treating a human is also disclosed. Further provided are methods of using oligonucleotide compounds for the inhibition of apolipoprotein B expression and for the treatment of diseases associated with apolipoprotein B activity. Examples of such diseases are various types of HDL / LDL cholesterol imbalance; dyslipidemia such as familial mixed hyperlipidemia (FCHL), acquired hyperlipidemia, hypercholestorolemia; statins Resistant hypercholesterolemia; coronary artery disease (CAD), coronary heart disease (CHD), atherosclerosis.

  The present invention further provides the use of a compound or complex as defined herein for the manufacture of a medicament for the treatment of abnormal levels of Apo-B100 or a disease or condition associated therewith.

  The present invention further provides a medicament comprising a compound or complex of the present invention for the treatment of abnormal levels of Apo-B100 or a disease or condition associated therewith.

  In one embodiment, the diseases and symptoms associated with abnormal levels of Apo-B100 can be selected from the group consisting of: atherosclerosis, hypercholesterolemia or hyperlipidemia.

  The present invention relates to a disease selected from atherosclerosis, hypercholesterolemia and hyperlipidemia, comprising the step of administering to a subject in need a pharmaceutical composition or complex as defined herein. Further provided are methods of treating a subject suffering from symptoms.

  The present invention relates to a pharmaceutical composition or complex as defined herein suffering from a medical condition selected from the group consisting of a subject, for example atherosclerosis, hypercholesterolemia or hyperlipidemia. There is further provided a method of down-regulating apolipoprotein B comprising administering to a subject.

Salt The oligomeric compound can be employed in various pharmaceutically acceptable salt forms. As used herein, the term refers to a salt that retains the desired biological activity of the LNA oligonucleotide and exhibits minimal undesirable toxic effects. Non-limiting examples of such salts can be formed with organic amino acids, with metal cations such as zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium, sodium, potassium, etc., or ammonia, It may be a base addition salt formed with a cation formed from N, N-dibenzylethylene-diamine, D-glucosamine, tetraethylammonium or ethylenediamine; or a combination, such as a zinc tannate salt.

  Such salts are formed from oligomeric compounds having a phosphordiester group and / or a phosphorothioate group, for example a salt with a suitable base. These salts are, for example, nontoxic metal salts obtained from metals of groups Ia, Ib, IIa and IIb of the periodic table of elements, in particular suitable alkali metal salts such as lithium, sodium or potassium salts, or alkali Includes earth metal salts such as magnesium or calcium salts. They further comprise zinc and ammonium salts and together with suitable organic amines, such as unsubstituted or hydroxyl-substituted mono-, di- or tri-alkyl amines, especially mono-, di- or tri-alkyl amines, or quaternary Along with ammonium compounds, for example N-methyl-N-ethylamine, diethylamine, triethylamine, mono-, bis- or tris- (2-hydroxy-lower alkyl) amines, such as mono-, bis- or tris- (2-hydroxyethyl) Amines, 2-hydroxy-tert-butylamine or tris (hydroxymethyl) methylamine, N, N-di-lower alkyl-N- (hydroxy-lower alkyl) amines such as N, N-dimethyl-N- (2-hydroxy Ethyl) amine or tri- (2-hydroxyethyl) amine, or N-methyl-D-glucamine, or a quaternary ammonium compound Both salts formed, including, for example, tetrabutylammonium salts. Lithium salts, sodium salts, magnesium salts, zinc salts or potassium salts are preferred, and sodium salts are particularly preferred.

Prodrug In one embodiment, the LNA oligonucleotide may be in the form of a prodrug. Oligonucleotides are negatively charged ions by virtue. Due to the lipophilic nature of the cell membrane, the uptake of oligonucleotides into cells is small compared to neutral or lipophilic counterparts. This polarity “hindrance” can be avoided by using a prodrug approach (eg Crooke, RM (1998) in Crooke, ST Antisense research and Application. Springer-Verlag, Berlin, Germany, vol. 131, pp 103-140). In this approach, the oligomeric compound is prepared in a protected manner such that the oligomeric compound is neutral when administered. These protecting groups are designed so that they can be removed when the LNA oligonucleotide is taken up by the cell. Examples of such protecting groups are S-acetylthioethyl (SATE) or S-pivaloylthioethyl (t-butyl-SATE). These protecting groups are nuclease resistant and are selectively removed intracellularly.

Aspects of the Invention The following aspects can be combined with the features of the invention referred to herein:

  1. an oligomeric compound comprising a total of 10-15 nucleobases, wherein the nucleobase sequence is present in SEQ ID NO: 1 and comprises at least 2 or at least 3 nucleotide analogues.

  2. If the nucleobase sequence is 5 ′ to 3 ′, i) region A: a sequence of 2-4 nucleotide analogues, ii) the following region B: a sequence of 6-11 nucleotides (eg DNA A compound of embodiment 1 comprising nucleotides), iii) followed by region C: a series of 2-4 nucleotide analogs, and optionally iv) 1 or 2 nucleotides (D).

  3. The compound of embodiment 2, wherein region A has a length of 2 nucleotide analogs.

  4. The compound of embodiment 2, wherein region A has a length of 3 nucleotide analogs.

  5. The compound of embodiment 2, wherein region A has a length of 4 nucleotide analogs.

  6. The compound of any of embodiments 2-5, wherein region C has a length of 2 nucleotide analogs.

  7. The compound of any of embodiments 2-5, wherein region C has a length of 3 nucleotide analogs.

  8. The compound of any of embodiments 2-5, wherein region C has a length of 4 nucleotide analogs.

  9. The compound of any of embodiments 2-8, wherein region B has a length of between 7 and 10 nucleotides (eg, DNA nucleotides), such as 8 or 9 nucleotides (eg, DNA nucleotides).

  10. A compound according to any of the preceding embodiments having a length of 12 to 15 nucleobases.

  11. The compound of embodiment 10, having a length of 12, 13, or 14 nucleobases.

12. The nucleobase sequence includes an internucleobase binding group selected from the group consisting of a phosphate group, a phosphodiester group, a phosphorothioate group, and a boranophosphate group, and the internucleoside bond is —OP (O) ₂ —O—. , —OP (O, S) —O—, The compound of any of the previous embodiments, which can be

  13. The compound of any of embodiments 1-12, wherein the internucleobase linking group between nucleobase units of the nucleobase sequence is independently selected from either phosphorothioate or phosphodiester linking groups.

  14. The compound of embodiment 12 or 13, wherein region A comprises at least one phosphodiester bond between two nucleotide analog units or between a nucleotide analog unit and a nucleotide unit.

  15. The compound of any of embodiments 12-14, wherein region C comprises at least one phosphodiester bond between two nucleotide analog units or between a nucleotide analog unit and a nucleotide unit.

  16. The compound of any of embodiments 12-15, wherein the internucleotide linkage between the nucleotides of region B is phosphorothioate.

  17. The compound of any of embodiments 12-16, wherein the internucleobase linkage between the 3 ′ nucleotide analog of A and the 5 ′ nucleotide of region B is phosphorothioate.

  18. The compound of any of embodiments 12-17, wherein the internucleobase linkage between the 3 ′ nucleotide of region B and the 5 ′ nucleotide analog of region C is phosphorothioate.

  19. The compound of any of embodiments 10-16 and 18, wherein the internucleobase linkage between the 3 ′ nucleotide analog of A and the 5 ′ nucleotide of region B is a phosphodiester.

  20. The compound of any of embodiments 10-17 and 19, wherein the internucleobase linkage between the 3 ′ nucleotide of region B and the 5 ′ nucleotide analog of region C is a phosphodiester.

  21. The compound of any of embodiments 12-20, wherein the internucleobase linkage between the two 5 ′ nucleotide analogs of region A is a phosphodiester.

  22. The compound of any of embodiments 12-21, wherein the internucleobase linkage between the two 3 ′ nucleotide analogs of region C is a phosphodiester.

  23. The compound of any of embodiments 12-22, wherein the internucleobase linkage between the two 3 ′ nucleotide analogs of region A is a phosphodiester.

  24. The compound of any of embodiments 12-23, wherein the internucleobase linkage between the two 5 ′ nucleotide analogs of region C is a phosphodiester.

  25. The compound of any of embodiments 12-24, wherein region A has a length of 4 nucleotide analogs and the internucleobase linkage between the two central nucleotide analogs of region A is a phosphodiester.

  26. The compound of any of embodiments 12-25, wherein region C has a length of 4 nucleotide analogs and the internucleobase linkage between the two central nucleotide analogs of region C is a phosphodiester.

  27. The compound of any of embodiments 10-26, wherein all internucleobase linkages between nucleotide analogs are phosphodiesters.

  28. The compound of any of embodiments 10-27, wherein all remaining internucleobase linkages are either phosphodiesters or phosphorothioates.

  29. The compound of embodiment 12, wherein all the internucleobase linkage groups are phosphorothioates.

  30. The compound of any of embodiments 1-29, wherein the nucleotide analog is independently selected from the group consisting of: 2′-O-alkyl-RNA monomer, 2′-amino-DNA monomer, 2′-fluoro -DNA monomers, LNA monomers, arabino nucleic acid (ANA) monomers, 2'-fluoro-ANA monomers, HNA monomers, INA monomers.

  31. The compound of embodiment 30, wherein the nucleotide analog is independently selected from the group consisting of 2′-MOE-RNA (2′-O-methoxyethyl-RNA), 2′fluoro-DNA, and LNA.

  32. The compound of embodiment 30 or 31, wherein at least one of said nucleotide analogs is a locked nucleic acid (LNA).

  33. Embodiments 30-32 wherein at least one of said nucleotide analogues is a 2′-MOE-RNA, eg 2, 3, 4, 5, 6, 7 or 8 2′-MOE-RNA nucleobase units Any compound.

  34. Any of embodiments 30-33, wherein at least one of said nucleotide analogs is 2'-fluoro DNA, eg 2, 3, 4, 5, 6, 7 or 8 2'-fluoro-DNA nucleobase units Some compounds.

  35. Of the nucleotide analogs, 2, 3, 4, 5, 6, 7 or 8 are LNAs, any of the remaining nucleotide analogs being a group of nucleotide analogs according to any of embodiments 26-30 A compound according to any of embodiments 30-34, which may be selected from:

  36. The compound of embodiment 32, wherein all nucleotide analogs are LNA.

  37. The compound of any of embodiments 30-36, wherein the LNA is selected from beta-D-oxy-LNA, alpha-L-oxy-LNA, beta-D-amino-LNA and beta-D-thio-LNA. .

  38. Any of aspects 1-37, wherein the nucleobase sequence is selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15. Some compounds.

  39. The compound of embodiment 38, selected from the group consisting of SEQ ID NOs: 17, 18, 19, 20, 21, 22, 23, 24 and 25.

  40. The compound of embodiment 38, selected from the group consisting of SEQ ID NOs: 26, 27, 28, 29, 30, 31, 32 and 33.

  41. The compound of embodiment 38, selected from the group consisting of SEQ ID NOs: 34, 35, 36, 37, 38, 39 and 40.

  42. A complex comprising a compound of any of embodiments 1-41 and at least one non-nucleotide or non-polynucleotide moiety covalently linked to said compound.

  43. The complex of embodiment 42, wherein said non-nucleotide or non-polynucleotide moiety consists of or comprises a sterol group, such as cholesterol.

  44. A pharmaceutical composition comprising a compound as defined in any of aspects 1-40 or a complex as defined in aspect 42 or 43, and a pharmaceutically acceptable diluent, carrier or adjuvant.

  45. The pharmaceutical composition of embodiment 44, further comprising at least one cholesterol lowering compound.

  46. said compound is a bile salt scavenging resin (e.g. cholestyramine, cholestipol and colesevelam hydrochloride), HMGCoA-reductase inhibitor (e.g. lovastatin, cerivastatin, pravastatin, atorvastatin, simvastatin and fluvastatin), nicotinic acid, Fibric acid derivatives (e.g. clofibrate, gemfibrozil, fenofibrate, bezafibrate, and ciprofibrate), probucol, neomycin, dextrothyroxine, plant stanol esters, cholesterol absorption inhibitors (e.g. ezetimibe), imputapide, bile acid transporter (apical) (apical) inhibitors of sodium-dependent bile acid transporters, regulators of liver CYP7a, estrogen replacement therapy (e.g., tamoxi E down), and anti-inflammatory agents (e.g., selected from the group consisting of glucocorticoids), pharmaceutical composition of embodiment 45.

  47. A compound or complex as defined in any of embodiments 1-43 for use as a medicament.

  48. Use of a compound or conjugate as defined in any of embodiments 1-43 for the manufacture of a medicament for the treatment of abnormal levels of Apo-B100 or a disease or condition associated therewith.

  49. A medicament comprising a compound or conjugate of any of embodiments 1-43 for the treatment of abnormal levels of Apo-B100 or a disease or condition associated therewith.

  50. The use or medicament of embodiment 48 or 49, wherein said abnormal level of Apo-B100 is associated with the presence of a medical condition selected from the group consisting of atherosclerosis, hypercholesterolemia or hyperlipidemia .

  51. A disease selected from atherosclerosis, hypercholesterolemia and hyperlipidemia comprising the step of administering to a subject in need thereof a pharmaceutical composition or complex as defined in any of embodiments 1-43 Or a method of treating a subject suffering from symptoms.

  52. A pharmaceutical composition or complex as defined in any of embodiments 1-43, suffering from a medical condition selected from the group consisting of a subject, for example atherosclerosis, hypercholesterolemia or hyperlipidemia A method of downregulating apolipoprotein B comprising the step of administering to a subject.

Examples Example 1: Synthesis of monomers
LNA monomer components and their derivatives were prepared using standard methods, eg, published procedures and references cited in WO07 / 031081.

Example 2: Oligonucleotide Synthesis Table 1 Oligonucleotide Compounds of the Invention In SEQ ID NOs: 16-40, uppercase letters indicate nucleotide analog units, and the subscript “s” indicates a phosphorothioate bond. The absence of “s” indicates a phosphodiester bond.

Example 3: Plasma Cholesterol Levels Plasma total cholesterol levels are measured using the Cometric Color Assay Cholesterol CP from ABX Pentra. Cholesterol is measured after enzymatic hydrolysis and oxidation. 21.5 μL of water was added to 1.5 μL of plasma. Add 250 μL of reagent and measure cholesterol content at a wavelength of 540 nM within 5 minutes. Measurements for each animal were repeated twice. Sensitivity and linearity were tested using a 2-fold diluted control compound (ABX Pentra N control). Relative cholesterol levels were determined by subtracting the background and expressed relative to the cholesterol levels in the plasma of mice treated with saline.

  Lipoproteins in plasma were separated from density-adjusted plasma into HDL and non-HDL fractions by ultracentrifugation. Non-HDL fractions mainly contain VLDL and LDL. Cholesterol content in each of the two fractions was analyzed as described for total cholesterol analysis using a commercial kit for total cholesterol from ABX Diagnostics, France.

Example 4: Measurement of mRNA levels
Antisense modulation of Apo-B100 expression can be assayed in a variety of ways known in the art. For example, Apo-B100 mRNA levels can be quantified, for example, by Northern blot analysis, competitive polymerase chain reaction (PCR), or real-time PCR. Currently, real-time quantitative PCR is preferred. RNA analysis can be performed on total cellular RNA or mRNA. Methods of RNA isolation and RNA analysis, such as Northern blot analysis, are routine in the art and are taught, for example, in Current Protocols in Molecular Biology, John Wiley and Sons.

  Real-time quantitative (PCR) can be conveniently achieved using the iQ Multi-Color Real Time PCR Detection System commercially available from BioRAD. Real-time quantitative PCR is a well-known technique in the art and is taught, for example, in Heid et al. Real time quantitative PCR, Genome Research (1996), 6: 986-994.

Example 5: Screening for oligonucleotides targeting ApoB-100 mRNA (administration of 3 * 5 mg / kg)
In this study, 5 mg / kg / dose was administered for 3 consecutive days (1 dose / day by intravenous infusion) and the animals were sacrificed 24 hours after the last dose. At the time of sacrifice, liver and retroorbital sinus blood were collected. Serum was prepared from blood for cholesterol analysis. RNA was isolated from the liver and ApoB-100 mRNA expression was measured.

  The effect of 3 doses of oligos of different lengths at 5 mg / kg / dose on ApoB-100 mRNA expression is shown in FIG. SEQ ID NO: 16 down-regulated ApoB-100 mRNA by about 25-30%, while 14-mer SEQ ID NO: 17 and 12-mer SEQ ID NO: 34 were equally much more potent-3 mg / kg 3 doses After that, ApoB-100 mRNA was down-regulated by about 75%.

  At the time of sacrifice on the third day, total cholesterol in the serum was measured (FIG. 2). Similar to the results from qPCR, the best or most potent effect was obtained with 12-mer SEQ ID NO: 34, followed by 14-mer SEQ ID NO: 17. 16-mer (SEQ ID NO: 16) reduced total cholesterol by approximately 18%.

Example 6: Dose response and duration of action of SEQ ID NO: 17 and SEQ ID NO: 34 in C57BL / 6 female mice In this study, three different concentrations (10, 15 and 25 mg / kg) of SEQ ID NO: 17 and SEQ ID NO: 34 were ApoB-100 mRNA expression and time of action on serum cholesterol levels were tested. SEQ ID NO: 17 and SEQ ID NO: 34 were given to C57BL / 6 female mice as a single dose of 10, 15 or 25 mg / kg. After administration, mice were sacrificed at different time points (1, 3, 5 and 8 days); liver and serum were tested for ApoB-100 mRNA expression, liver oligonucleotide concentration, and cholesterol and ALT, respectively.

Analysis of down-regulation of target mRNA The liver collected at the time of sacrifice was analyzed for ApoB-100 mRNA expression by qPCR. Data were normalized to Gapdh and expressed relative to data obtained with saline administration. A single dose of 10, 15, or 25 mg / kg of SEQ ID NO: 17 or SEQ ID NO: 34 was very effective in down-regulating ApoB-100 mRNA in the liver (Figure 3). At 24 hours post-dose, 90-95% down-regulation was obtained with SEQ ID NO: 34, whereas administration of SEQ ID NO: 17 resulted in 70-85% lower ApoB mRNA levels than the saline control group.

Serum cholesterol
Serum used to measure cholesterol was collected at the time of sacrifice. Serum total cholesterol decreased by 25-40% 24 hours after administration of SEQ ID NO: 17, and administration of SEQ ID NO: 34 resulted in a 40-55% decrease in total cholesterol. On day 3, total cholesterol levels were further reduced: SEQ ID NO: 17 resulted in a 70-90% reduction in a dose-dependent manner after administration of 10, 15 or 25 mg / kg. SEQ ID NO: 34 reduced total cholesterol by 90-95% compared to the saline control group. On days 5-8, total cholesterol levels increased in all groups except those administered SEQ ID NO: 17 at 10 mg / kg (Figure 4).

Example 7: Dose response and duration of action of SEQ ID NO: 17 and SEQ ID NO: 34 in C57BL / 6 female mice To find ED50 values for cholesterol, single doses of SEQ ID NO: 17 and SEQ ID NO: 34 were administered at different concentrations with C57BL / Administered to 6J mice. Since we have previously experienced that the maximum effect of a single dose is not always achieved 24 hours after administration, the time of action was also included in this study. In Example 6, we completely downregulated ApoB-100 mRNA after administration of 10, 15 or 25 mg / kg of SEQ ID NO: 34 and 25 mg / kg of SEQ ID NO: 17. Therefore, we chose lower concentrations (1, 2.5 and 5 mg / kg) in this study.

Analysis of down-regulation of target mRNA The liver collected at the time of sacrifice was analyzed for ApoB-100 mRNA expression by qPCR. Data were normalized to Gapdh and expressed relative to data obtained with saline administration. A single dose of 10, 15 or 25 mg / kg of SEQ ID NO: 17 or SEQ ID NO: 34 was very effective in down-regulating ApoB-100 mRNA in the liver (Figure 5). A single dose of 1, 2.5 or 5 mg / kg of SEQ ID NO: 17 resulted in a dose-dependent down-regulation of ApoB-100 mRNA for 5 days. Similar results were obtained with SEQ ID NO: 34. On day 8, both oligonucleotides resulted in similar ApoB-100 mRNA expression, ie, a 75% reduction after administration of 2.5 mg / kg SEQ ID NO: 34 and 5 mg / kg SEQ ID NO: 17. On day 16, all groups had mRNA levels, except after administration of 5 mg / kg of SEQ ID NO: 34, which resulted in 75% ApoB-100 mRNA downregulation similar to days 5 and 8. Increased again.

Serum cholesterol
Serum was collected at the time of sacrifice and used to measure cholesterol. Serum total cholesterol levels reflected ApoB-100 mRNA expression; best effect 5 days after administration of SEQ ID NO: 17 at 1 and 2.5 mg / kg, and 3, 5 and 8 after administration of 5 mg / kg Dose-dependent decrease with similar effect on day (50% decrease). The dose-dependent effect had the best effect (70% decrease) 3 days after administration of 5 mg / kg, followed by an increase in cholesterol levels (60% decrease on day 8 and 45% decrease on day 16) ) And was also obtained after administration of SEQ ID NO: 34. However, cholesterol levels in the group administered SEQ ID NO: 34 did not follow mRNA reduction in the groups administered 2.5 and 5 mg / kg. For example, administration of 5 mg / kg resulted in about 75% down-regulation of ApoB-100 mRNA on days 5-16, while cholesterol levels after administration of 2.5 mg / kg and 5 mg / kg were 3 days. From day to day 16, it increased from a 70% decrease to a 45% decrease.

Example 8: Screening for oligonucleotides targeting ApoB-100 mRNA (3 * 1 or 5 mg / kg administered for 3 consecutive days by intravenous infusion)
Three LNA antisense oligonucleotides, all targeting ApoB mRNA, 12-mer SEQ ID NO: 34, 13-mer SEQ ID NO: 26 and 14-mer SEQ ID NO: 17 at 1.0 or 5.0 mg / kg (0 The effects on ApoB-100 mRNA were tested on different days after a single dose on the day).

Analysis of down-regulation of target mRNA The liver collected at the time of sacrifice was analyzed for ApoB-100 mRNA expression by qPCR. Data were normalized to Gapdh and expressed relative to data obtained with saline administration. Administration of 3 * 1 or 5 mg / kg of SEQ ID NO: 34, SEQ ID NO: 17 or SEQ ID NO: 26 was very effective in down-regulating ApoB-100 mRNA in the liver (FIG. 7). Administration of 1 mg / kg of SEQ ID NO: 34 or SEQ ID NO: 26 down-regulated ApoB-100 mRNA by 60%, and administration of 5 mg / kg resulted in a similar 90% down-regulation for both compounds. SEQ ID NO: 17 administered at 3 * 1 mg / kg / dose or 5 mg / kg / dose down-regulated target mRNA by 50% and 70%, respectively.

Serum cholesterol
Serum blood was collected at the time of sacrifice and used to measure cholesterol. Similar to the results for mRNA expression, SEQ ID NO: 34 and SEQ ID NO: 26 produced similar results: 60% reduction after administration of 3 * 1 mg / kg and after administration of 3 * 5 mg / kg / dose Approximately 85-90% decrease. SEQ ID NO: 17 was slightly less potent and reduced serum cholesterol by 40% and 70% after administration of 3 * 1 or 5 mg / kg / dose, respectively.

Example 9: Dose regimen and potency of SEQ ID NO: 26 in C57BL / 6J female mice In this study, three different concentrations (1, 2.5 and 5 mg / kg) of SEQ ID NO: 26 were converted to plasma by ultracentrifugation. The lipoprotein fraction isolated from was tested for potency against total cholesterol and non-HDL and HDL cholesterol. SEQ ID NO: 26 was given to C57BL / 6J female mice by intravenous infusion at 1, 2.5 or 5 mg / kg for 6 weeks, either once a week or once every two weeks. Mice were sacrificed 1 week after the last dose (day 42 for all groups). Serum was collected weekly and analyzed for total cholesterol and non-HDL and HDL cholesterol.

Plasma cholesterol / total cholesterol Plasma used to measure cholesterol was collected once weekly and at the time of sacrifice.

  The group administered at 2.5 and 5 mg / kg / dose reached a steady state with a total cholesterol reduction of 60% after 14 days, so once weekly administration causes the oligonucleotide to accumulate in the liver So I thought. The 1 mg / kg dose resulted in a steady state with a 40% total cholesterol reduction after 28 days. At a dose of once every two weeks, a steady-state steady state with a 60% reduction in total cholesterol was achieved after administration of 5 mg / kg / dose for 28 days (after 2 doses). In the groups dosed at 1 and 2.5 mg / kg, total cholesterol fluctuations continued until day 35 after the first dose, but the fluctuations decreased with time. And steady state will probably be established with time even at these concentrations. For 1 and 2.5 mg / kg / dose, the reduction in total cholesterol varied between 20-30% and 40-50% from day 28 to day 35 (Figures 9A and B), respectively. .

Non-HDL and HDL cholesterol Plasma was separated into HDL and non-HDL fractions and analyzed separately for cholesterol content, between once a week (Figures 10A and B) and once every two weeks The difference was more clearly illuminated than when the plasma samples were analyzed directly for total cholesterol content. Figures 11A and B show that once every two weeks of usage resulted in a decrease in non-HDL cholesterol levels almost comparable to the effect of weekly infusions, whereas once every two weeks usage had more impact on HDL cholesterol. Shows that it was calm. It is noteworthy that an infusion once every two weeks was sufficient to reduce non-HDL cholesterol and that it provided beneficial lipoprotein properties with higher HDL cholesterol than weekly infusions.

Claims (22)

  An oligomeric compound comprising a total of 10-15 nucleobases, wherein the nucleobase sequence corresponds to a continuous subsequence present in SEQ ID NO: 1 and comprises at least 2 or at least 3 nucleotide analogues.   The sequence of the nucleobase is 5 ′ to 3 ′, i) region A: a sequence of 2-4 nucleotide analogues, ii) the following region B: a sequence of 6-11 nucleotides (eg DNA nucleotides) ), Iii) Subsequent region C: A compound according to claim 1, comprising a stretch of 2-4 nucleotide analogues, and optionally iv) 1 or 2 nucleotides (D).   12. A compound according to any one of the preceding claims having a nucleobase length of 12-15.   4. A compound according to claim 3, having a length of 12, 13 or 14 nucleobases.   5. A compound according to any one of claims 1-4, wherein the internucleobase linking group between nucleobase units of the nucleobase sequence is independently selected from either phosphorothioate or phosphodiester linking groups.   Nucleotide analogs are 2'-O-alkyl-RNA monomer, 2'-amino-DNA monomer, 2'-fluoro-DNA monomer, LNA monomer, arabino nucleic acid (ANA) monomer, 2'-fluoro-ANA monomer, HNA The compound according to any one of claims 1 to 5, which is independently selected from the group consisting of a monomer and an INA monomer.   7. A compound according to claim 6, wherein at least one of said nucleotide analogues is a locked nucleic acid (LNA).   7. A compound according to claim 6 wherein all nucleotide analogues are LNA.   The LNA according to any one of claims 6-8, wherein the LNA is selected from beta-D-oxy-LNA, alpha-L-oxy-LNA, beta-D-amino-LNA and beta-D-thio-LNA. The described compound.   The compound according to any one of claims 1 to 9, wherein the nucleobase sequence is selected from the group consisting of SEQ ID NO: 5, 9 or 13.   From SEQ ID NOs: 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 and 40 11. The compound of claim 10, selected from the group consisting of: and / or the group consisting of SEQ ID NOs: 41-49.   12. A compound according to claim 11 selected from the group consisting of SEQ ID NOs: 16, 17, 26 and 34.   13. A compound according to claim 12, which is SEQ ID NO: 26.   14. A complex comprising a compound according to any one of claims 1-13 and at least one non-nucleotide or non-polynucleotide moiety covalently bound to the compound.   A pharmaceutical composition comprising a compound as defined in any one of claims 1-13 or a complex as defined in claim 14 and a pharmaceutically acceptable diluent, carrier or adjuvant.   14. A compound or complex as defined in any one of claims 1-13 for use as a medicament.   14. A compound as defined in any one of claims 1-13 or as defined in claim 14 for the manufacture of a medicament for the treatment of abnormal levels of Apo-B100 or a disease or condition associated therewith. Use of complex.   15. A medicament comprising a compound according to any one of claims 1-13 or a complex according to claim 14 for the treatment of abnormal levels of Apo-B100 or a disease or condition associated therewith.   Use according to claim 17 or 18, wherein the abnormal level of Apo-B100 is associated with the presence of a medical condition selected from the group consisting of atherosclerosis, hypercholesterolemia or hyperlipidemia Medicine.   A disease selected from atherosclerosis, hypercholesterolemia and hyperlipidemia comprising administering to a subject in need a pharmaceutical composition or complex as defined in any one of the preceding claims Or a method of treating a subject suffering from symptoms.   A pharmaceutical composition or complex as defined in any one of claims 1-14 for a medical condition selected from the group of subjects, for example atherosclerosis, hypercholesterolemia or hyperlipidemia A method of downregulating apolipoprotein B comprising the step of administering to an afflicted subject.   ApoB in a cell expressing ApoB, comprising administering to the cell the compound of any one of claims 1-13 or the complex of claim 14 to downregulate apolipoprotein B (ApoB) mRNA. How to control down.

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